Our long term goal is to determine how signals which are generated by muscarinic receptor-activated inositol phospholipid metabolism are involved in activation of airway smooth muscle function. We also want to learn how production of these signals is controlled or modulated. Our studies currently are performed on porcine trachealis muscle and cultured human ~hypersensitive~ airway muscle. In the present work, we will emphasize investigation of two key enzymes involved in this signal transduction system; inositol phospholipid-specific phospholipase C (PLC), and phosphatidylinositol-4-phosphate 5-kinase (PI(4)P 5-kinase). For PLC, our specific aims are (a) to determine how this enzyme is activated and controlled during muscarinic stimulation; (b) to determine if there is feedback modulation exerted by increases in cytosolic Ca2+ concentration; to determine if phosphatidylinositol transfer protein is involved in delivering substrate to this enzyme. The specific aim in studies of cultured human airway smooth muscle which have been treated with TNFa is to determine why these muscles produce more Ins (1,4,5) P3 when activated with bradykinin than do cells which have not been treated with TNFa. In these investigations we use intact airway smooth muscle, subcellular fractions, and cultured human airway smooth muscle cells.